PRODUCTION OF SOLUBLE SOY PROTEIN PRODUCT FROM SOY PROTEIN MICELLAR MASS (&#34;S200Ca&#34;)

ABSTRACT

A soy protein product having a protein content of at least 60 wt % (N×6.25) d.b., preferably an isolate having a protein content of at least about 90 wt % (N×6.25) d.b., is formed from the supernatant from the precipitation of a soy protein micellar mass. A calcium salt or other divalent salt is added to the supernatant, before concentration, after initial concentration or after final concentration, to provide a conductivity of about 2 to about 30 mS. Precipitate is removed from the resulting solution and the pH of the clear soy protein solution is optionally adjusted to about 1.5 to about 4.4. The optionally pH-adjusted clear solution is concentrated to a concentration of about 50 to about 400 g/L and the clear concentrated protein solution is optionally diafiltered prior to drying. The soy protein product is soluble in acidic media and produces transparent, heat stable solutions at low pH values and, therefore, may be used for protein fortification of soft drinks and sports drinks.

REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC 119(e) from U.S.Provisional Patent Application Nos. 61/202,055 filed Jan. 26, 2009 and61/272,289 filed Sep. 8, 2009.

FIELD OF INVENTION

The invention relates to the production of soybean protein products.

BACKGROUND TO THE INVENTION

In U.S. Provisional Patent Applications Nos. 61/107,112 (7865-373) filedOct. 21, 2008, 61/193,457 (7865-374) filed Dec. 2, 2008, 61/202,070(7865-376) filed Jan. 26, 2009, 61/202,553 filed Mar. 12, 2009(7865-383), 61/213,717 (7865-389) filed Jul. 7, 2009, 61/272,241 filedSep. 3, 2009 and U.S. patent application Ser. No. 12/603,087 filed Oct.21, 2009 the disclosures of which are incorporated herein by reference,there is described the preparation of a soy protein product, preferablya soy protein isolate, which is completely soluble and is capable ofproviding transparent and heat stable solutions at low pH values. Thissoy protein product may be used for protein fortification of, inparticular, soft drinks and sports drinks, as well as other acidicaqueous systems, without precipitation of protein. The soy proteinproduct is produced by extracting a soy protein source with aqueouscalcium chloride solution at natural pH, optionally diluting theresulting aqueous soy protein solution, adjusting the pH of the aqueoussoy protein solution to a pH of about 1.5 to about 4.4, preferably about2.0 to about 4.0, to produce an acidified clear soy protein solution,which may be optionally concentrated and/or diafiltered before drying.

SUMMARY OF THE INVENTION

It has now been found that process streams derived from theprecipitation of a soy protein micellar mass may be further processed toprovide soy protein products having a protein content of at least about60 wt % (N×6.25) d.b. that are soluble in acidic media and producetransparent, heat stable solutions at low pH values, and, thereforewhich may be used for protein fortification of, in particular, softdrinks and sports drinks, as well as other aqueous systems, withoutprecipitation of protein. The soy protein product is preferably anisolate having a protein content of at least about 90 wt %, preferablyat least about 100 wt % (N×6.25) d.b.

In accordance with one aspect of the present invention, there isprovided a process of preparing a soy protein product having a proteincontent of at least about 60 wt % (N×6.25) on a dry weight basis, whichcomprises:

adding calcium salt or other divalent salt, preferably calcium chloride,to supernatant from the precipitation of a soy protein micellar mass toprovide a conductivity of about 2 mS to about 30 mS, preferably about 8to about 15 mS,

removing precipitated phytate material from the resulting solution toleave a clear solution,

optionally adjusting the pH of the clear solution to about 1.5 to about4.4, preferably about 2.0 to about 4.0, such as by the addition ofhydrochloric acid,

concentrating the optionally pH-adjusted clear solution to a proteincontent of about 50 to about 400 g/L, preferably about 100 to about 250g/L to produce a clear concentrated soy protein solution,

optionally diafiltering the clear soy protein solution, before or aftercomplete concentration, such as with about 2 to about 40 volumes ofwater, preferably about 5 to about 25 volumes of water,

optionally effecting a colour removal step, such as a granular activatedcarbon treatment, and

drying the concentrated protein solution.

The supernatant may be partially concentrated to an intermediateconcentration prior to addition of the calcium salt. The precipitatewhich forms is removed and the resulting solution is optionallyacidified as described above, further concentrated to the finalconcentration and then optionally diafiltered and dried.

Alternatively, the supernatant first may be concentrated to the finalconcentration, the calcium salt is added to the concentratedsupernatant, the resulting precipitate is removed and the solution isoptionally acidified and then optionally diafiltered and dried.

It is an option in the above-described procedures to omit theacidification and effect processing of the solution at natural pH. Inthis option calcium salt is added to supernatant, partially concentratedsupernatant or concentrated supernatant to form a precipitate which isremoved. The resulting solution then is processed as described abovewithout the acidification step.

Where the supernatant is partially concentrated prior to the addition ofthe calcium salt and fully concentrated after removal of theprecipitate, the supernatant is first concentrated to a proteinconcentration of about 50 g/L or less, and, after removal of theprecipitate, then is concentrated to a concentration of about 50 toabout 400 g/L, preferably about 100 to about 250 g/L.

The soy protein product preferably is an isolate having a proteincontent of at least about 90 wt %, preferably at least about 100 wt %(N×6.25) d.b.

In another aspect of the invention, we have found that an equivalentproduct may be produced from soy by the processing of soy proteinsolution from sodium salt extraction of the soy protein source material,by concentrating the soy protein solution, optionally diafiltering theconcentrated soy protein solution, optionally adjusting the pH of thesolution to about 2 to about 4, and drying the acidified solution.According to this aspect of the present invention, there is provided aprocess of preparing a soy protein product having a protein content ofat least about 60 wt % (N×6.25) dry weight, which comprises:

extracting a soy protein source to solubilize soy protein in the sourcematerial and to form an aqueous soy protein solution having a pH ofabout 5 to about 7,

concentrating the aqueous soy protein solution to a concentration ofabout 50 to about 400 g/L to form a concentrated soy protein isolate,

optionally diafiltering the soy protein solution, before or aftercomplete concentration thereof,

optionally adjusting the pH of the concentrated and diafiltered soyprotein solution to about 2 to about 4 to provide a clear acidified soyprotein solution, and

drying the soy protein solution.

The soy protein product preferably is an isolate having a proteincontent of at least about 90 wt %, preferably at least about 100 wt %(N×6.25) d.b.

It has also been found that soy protein isolate formed as a proteinmicellar mass and soy protein isolate derived from supernatant fromprotein micellar mass precipitation are soluble in acidic media and maybe used to provide aqueous solutions of acceptable clarity.

While the present invention refers mainly to the production of soyprotein isolates, it is contemplated that soy protein products of lesserpurity may be provided having similar properties to the soy proteinisolates. Such lesser purity products may have a protein concentrationof at least about 60% by weight (N×6.25) d.b.

The novel soy protein products of the invention can be blended withpowdered drinks for the formation of aqueous soft drinks or sportsdrinks by dissolving the same in water. Such blend may be a powderedbeverage.

The soy protein products provided herein may be provided as an aqueoussolution thereof having a high degree of clarity at acid pH values andwhich is heat stable at these pH values.

In another aspect of the present invention, there is provided an aqueoussolution of the soy product provided herein which is heat stable at lowpH. The aqueous solution may be a beverage, which may be a clearbeverage in which the soy protein product is completely soluble andtransparent or an opaque beverage in which the soy protein product doesnot increase the opacity.

The soy protein products produced according to the processes herein lackthe characteristic beany flavour of soy protein isolate and aresuitable, not only for protein fortification of acidic media, but may beused in a wide variety of conventional applications of protein isolates,including but not limited to protein fortification of processed foodsand beverages, emulsification of oils, as a body former in baked goodsand foaming agent in products which entrap gases. In addition, the soyprotein product may be formed into protein fibres, useful in meatanalogs, and may be used as an egg white substitute or extender in foodproducts where egg white is used as a binder. The soy protein productmay be used in nutritional supplements. Other uses of the soy proteinproduct are in pet foods, animal feed and in industrial and cosmeticapplications and in personal care products.

GENERAL DESCRIPTION OF THE INVENTION

The initial step of the process of providing the soy protein productinvolves solubilizing soy protein from a soy protein source. The soyprotein source may be soybeans or any soy product or by-product derivedfrom the processing of soybeans including but not limited to soy meal,soy flakes, soy grits and soy flour. The soy protein source may be usedin the full fat form, partially defatted form or fully defatted form.Where the soy protein source contains an appreciable amount of fat, anoil-removal step generally is required during the process. The soyprotein recovered from the soy protein source may be the proteinnaturally occurring in soybean or the proteinaceous material may be aprotein modified by genetic manipulation but possessing characteristichydrophobic and polar properties of the natural protein.

Protein solubilization may be effected by using a food grade sodium saltsolution such as a solution of food grade sodium chloride. Where the soyprotein isolate is intended for non-food uses, non-food-grade chemicalsmay be used. Other monovalent salts also may be used, such as potassiumchloride. As the concentration of the salt solution increases, thedegree of solubilization of protein from the soy protein sourceinitially increases until a maximum value is achieved. Any subsequentincrease in salt concentration does not increase the total proteinsolubilized. The concentration of the salt solution which causes maximumprotein solubilization varies depending on the salt concerned. Thechoice of concentration of the sodium salt solution is also influencedby the proportion of protein desired to be obtained by the micellarroute. Higher salt concentrations, preferably about 0.5 M to about 1.0M, generally result in more protein micellar mass upon dilution of theconcentrated soy protein solution into cold water. The extraction may becarried out with a sodium chloride solution of higher concentration, oralternatively, the extraction can be carried out with a solution of lessthan 0.5 M sodium chloride, for example, 0.10 M or 0.15 M sodiumchloride, and then additional salt may be added to the soy proteinsolution after removal of the soy protein source.

In a batch process, the salt solubilization of the protein is effectedat a temperature of from about 1° C. to about 100° C., preferably about15° C. to about 35° C., preferably accompanied by agitation to decreasethe solubilization time, which is usually about 1 to about 60 minutes.It is preferred to effect the solubilization to extract substantially asmuch protein from the soy protein source as is practicable, so as toprovide an overall high product yield.

In a continuous process, the extraction of the protein from the soyprotein source is carried out in any manner consistent with effecting acontinuous extraction of protein from the soy protein source. In oneembodiment, the soy protein source is continuously mixed with a foodgrade salt solution and the mixture is conveyed through a pipe orconduit having a length and at a flow rate for a residence timesufficient to effect the desired extraction in accordance with theparameters described herein. In such continuous procedure, the saltsolubilization step is effected rapidly, in a time of up to about 10minutes, preferably to effect solubilization to extract substantially asmuch protein from the soy protein source as is practicable. Thesolubilization in the continuous procedure is effected at temperaturesbetween about 1° C. and about 100° C., preferably between about 15° C.and about 35° C.

The extraction may be carried out at the natural pH of the soy proteinsource/salt solution system, generally about 5 to about 7.Alternatively, the pH of the extraction may be adjusted to any desiredvalue within the range of about 5 to about 7 for use in the extractionstep by the use of any convenient acid, usually hydrochloric acid, oralkali, usually sodium hydroxide, as required.

The concentration of the soy protein source in the food grade saltsolution during the solubilization step may vary widely. Typicalconcentration values are about 5 to about 15% w/v.

The protein extraction step with the aqueous salt solution has theadditional effect of solubilizing fats which may be present in the soyprotein source, which then results in the fats being present in theaqueous phase.

The protein solution resulting from the extraction step generally has aprotein concentration of about 5 to about 50 g/L, preferably about 10 toabout 50 g/L.

The aqueous salt solution may contain an antioxidant. The antioxidantmay be any convenient antioxidant, such as sodium sulfite or ascorbicacid. The quantity of antioxidant employed may vary from about 0.01 toabout 1 wt % of the solution, preferably about 0.05 wt %. Theantioxidant serves to inhibit the oxidation of any phenolics in theprotein solution.

The aqueous phase resulting from the extraction step then may beseparated from the residual soy protein source, in any convenientmanner, such as by employing a decanter centrifuge, followed by disccentrifugation and/or filtration to remove residual soy protein sourcematerial. The separated residual soy protein source may be dried fordisposal. Alternatively, the separated residual soy protein source maybe processed to recover some residual protein, such as by a conventionalisoelectric precipitation procedure or any other convenient procedure torecover such residual protein.

Where the soy protein source contains significant quantities of fat, asdescribed in U.S. Pat. Nos. 5,844,086 and 6,005,076, assigned to theassignee hereof and the disclosures of which are incorporated herein byreference, then the defatting steps described therein may be effected onthe separated aqueous protein solution. Alternatively, defatting of theseparated aqueous protein solution may be achieved by any otherconvenient procedure.

The aqueous soy protein solution may be treated with an adsorbent, suchas powdered activated carbon or granulated activated carbon, to removecolour and/or odour compounds. Such adsorbent treatment may be carriedout under any convenient conditions, generally at the ambienttemperature of the separated aqueous protein solution. For powderedactivated carbon, an amount of about 0.025% to about 5% w/v, preferablyabout 0.05% to about 2% w/v, is employed. The adsorbing agent may beremoved from the soy protein solution by any convenient means, such asby filtration.

As an alternative to extracting the soy protein source with an aqueoussalt solution, such extraction may be made using water alone. Where suchalternative is employed, then the salt, in the concentrations discussedabove, may be added to the protein solution after separation from theresidual soy protein source. When a first fat removal step is carriedout, the salt generally is added after completion of such operations.

Another alternative procedure is to extract the soy protein source withthe food grade salt solution at a relatively high pH value above about7, generally up to about 11. The pH of the extraction system may beadjusted to the desired alkaline value by the use of any convenientfood-grade alkali, such as aqueous sodium hydroxide solution.Alternatively, the soy protein source may be extracted with the saltsolution at a relatively low pH below about pH 5, generally down toabout pH 3. The pH of the extraction system may be adjusted to thedesired acidic value by the use of any convenient food grade acid suchas hydrochloric or phosphoric acid. Where such alternative is employed,the aqueous phase resulting from the soy protein source extraction stepthen is separated from the residual soy protein source, in anyconvenient manner, such as by employing decanter centrifugation,followed by disc centrifugation and/or filtration to remove residual soyprotein source. The separated residual soy protein source may be driedfor disposal or further processed to recover residual protein, asdiscussed above.

The aqueous soy protein solution resulting from the high or low pHextraction step then is pH adjusted to the range of about 5 to about 7,as discussed above, prior to further processing as discussed below. SuchpH adjustment may be effected using any convenient acid, such ashydrochloric acid, or alkali, such as sodium hydroxide, as appropriate.If necessary, the protein solution may be clarified by any convenientprocedure such as centrifugation or filtration after the pH adjustmentand prior to further processing.

If of adequate purity, the resulting aqueous soy protein solution may bedirectly dried to produce a soy protein product. To decrease theimpurities content, the aqueous soy protein solution may be processedprior to drying.

The aqueous soy protein solution may be concentrated to increase theprotein concentration thereof while maintaining the ionic strengththereof substantially constant. Such concentration generally is effectedto provide a concentrated protein solution having a proteinconcentration of about 50 g/L to about 400 g/L, preferably about 100 toabout 250 g/L.

The concentration step may be effected in any convenient mannerconsistent with batch or continuous operation, such as by employing anyconvenient selective membrane technique, such as ultrafiltration ordiafiltration, using membranes, such as hollow-fibre membranes orspiral-wound membranes, with a suitable molecular weight cut-off, suchas about 3,000 to about 1,000,000 daltons, preferably about 5,000 toabout 100,000 daltons, having regard to differing membrane materials andconfigurations, and, for continuous operation, dimensioned to permit thedesired degree of concentration as the aqueous protein solution passesthrough the membranes.

As is well known, ultrafiltration and similar selective membranetechniques permit low molecular weight species to pass through themembrane while preventing higher molecular weight species from so doing.The low molecular weight species include not only the ionic species ofthe food grade salt but also low molecular weight materials extractedfrom the source material, such as, carbohydrates, pigments, lowmolecular weight proteins and anti-nutritional factors, such as trypsininhibitors, which are themselves low molecular weight proteins. Themolecular weight cut-off of the membrane is usually chosen to ensureretention of a significant proportion of the protein in the solution,while permitting contaminants to pass through having regard to thedifferent membrane materials and configurations.

The protein solution may be subjected to a diafiltration step, before orafter complete concentration, preferably using an aqueous salt solutionof the same molarity and pH as the extraction solution. If a reductionin the salt content of the retentate is desired, the diafiltrationsolution employed may be an aqueous salt solution at the same pH butlower salt concentration than the extraction solution. However, the saltconcentration of the diafiltration solution must be chosen so that thesalt level in the retentate remains sufficiently high to maintain thedesired protein solubility. Diafiltration may be effected using fromabout 2 to about 40 volumes of diafiltration solution, preferably about5 to about 25 volumes of diafiltration solution. In the diafiltrationoperation, further quantities of contaminants are removed from theaqueous protein solution by passage through the membrane with thepermeate. The diafiltration operation may be effected until nosignificant further quantities of contaminants or visible colour arepresent in the permeate. If the retentate is to be dried without furtherprocessing, according to one aspect of the present invention, thendiafiltration may be conducted until the retentate has been sufficientlypurified so as, when dried, to provide the desired proteinconcentration, preferably to provide an isolate with a protein contentof at least about 90 wt % (N×6.25) on a dry basis. Such diafiltrationmay be effected using the same membrane as for the concentration step.However, if desired, the diafiltration step may be effected using aseparate membrane with a different molecular weight cut-off, such as amembrane having a molecular weight cut-off in the range of about 3,000to about 1,000,000 daltons, preferably about 5,000 to about 100,000daltons, having regard to different membrane materials andconfiguration.

The concentration step and the diafiltration step may be effected hereinin such a manner that the soy protein product subsequently recovered bydrying the concentrated and diafiltered retentate contains less thanabout 90 wt % protein (N×6.25) d.b., such as at least about 60 wt %protein (N×6.25) d.b. By partially concentrating and/or partiallydiafiltering the aqueous soy protein solution, it is possible to onlypartially remove contaminants. This protein solution may then be driedto provide a soy protein product with lower levels of purity. The soyprotein product is still able to produce clear protein solutions underacidic conditions.

An antioxidant may be present in the diafiltration medium during atleast part of the diafiltration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit theoxidation of any phenolics present in the concentrated soy proteinsolution.

The concentration step and the optional diafiltration step may beeffected at any convenient temperature, generally about 2° to about 60°C., preferably about 20° to about 35° C., and for the period of time toeffect the desired degree of concentration and diafiltration. Thetemperature and other conditions used to some degree depend upon themembrane equipment used to effect the membrane processing, the desiredprotein concentration of the solution and the efficiency of the removalof contaminants to the permeate.

There are two main trypsin inhibitors in soy, namely the Kunitzinhibitor, which is a heat-labile molecule with a molecular weight ofapproximately 21,000 Daltons, and the Bowman-Birk inhibitor, a moreheat-stable molecule with a molecular weight of about 8,000 Daltons. Thelevel of trypsin inhibitor activity in the final soy protein isolate canbe controlled by manipulation of various process variables.

For example, the concentration and/or diafiltration steps may beoperated in a manner favorable for removal of trypsin inhibitors in thepermeate along with the other contaminants. Removal of the trypsininhibitors is promoted by using a membrane of larger pore size, such asabout 30,000 to about 1,000,000 Da, operating the membrane at elevatedtemperatures, such as about 30 to about 60° C. and employing greatervolumes of diafiltration medium, such as about 20 to about 40 volumes.

Further, a reduction in trypsin inhibitor activity may be achieved byexposing soy materials to reducing agents that disrupt or rearrange thedisulfide bonds of the inhibitors. Suitable reducing agents includesodium sulfite, cysteine and N-acetylcysteine.

The addition of such reducing agents may be effected at various stagesof the overall process. The reducing agent may be added with the soyprotein source material in the extraction step, may be added to theclarified aqueous soy protein solution following removal of residual soyprotein source material, may be added to the concentrated proteinsolution before or after diafiltration or may be dry blended with thedried soy protein product. The addition of the reducing agent may becombined with the membrane processing steps, as described above.

If it is desired to retain active trypsin inhibitors in the concentratedprotein solution, this can be achieved by utilizing a concentration anddiafiltration membrane with a smaller pore size, operating the membraneat lower temperatures, employing fewer volumes of diafiltration mediumand not employing a reducing agent.

The concentrated and optionally diafiltered protein solution may besubject to a further defatting operation, if required, as described inU.S. Pat. Nos. 5,844,086 and 6,005,076. Alternatively, defatting of theconcentrated and optionally diafiltered protein solution may be achievedby any other convenient procedure.

The concentrated and diafiltered aqueous protein solution may be treatedwith an adsorbent, such as powdered activated carbon or granulatedactivated carbon, to remove colour and/or odour compounds. Suchadsorbent treatment may be carried out under any convenient conditions,generally at the ambient temperature of the concentrated proteinsolution. For powdered activated carbon, an amount of about 0.025% toabout 5% w/v, preferably about 0.05% to about 2% w/v, is employed. Theadsorbent may be removed from the soy protein solution by any convenientmeans, such as by filtration.

The concentrated and optionally diafiltered soy protein solutionresulting from the optional defatting and optional adsorbent treatmentstep may be subjected to a pasteurization step to reduce the microbialload. Such pasteurization may be effected under any desiredpasteurization conditions. Generally, the concentrated and optionallydiafiltered protein solution is heated to a temperature of about 55° toabout 70° C., preferably about 60° to about 65° C., for about 30 secondsto about 60 minutes, preferably about 10 minutes to about 15 minutes.The pasteurized, concentrated protein solution then may be cooled forfurther processing as described below, preferably to a temperature ofabout 25° to about 40° C.

In accordance with one aspect of the present invention, the concentratedand diafiltered soy protein solution is dried to yield the soy proteinproduct. Alternatively, the concentrated and diafiltered soy proteinsolution may be adjusted in pH to a pH of about 2.0 to about 4.0,preferably about 2.9 to about 3.2. The pH adjustment may be effected inany convenient manner, such as by addition of hydrochloric acid orphosphoric acid. The resulting acidified soy protein solution then isdried. As a further alternative, the pH adjusted soy protein solutionmay be subjected to a heat treatment to inactivate heat labileanti-nutritional factors, such as the trypsin inhibitors mentionedabove. Such a heating step also provides the additional benefit ofreducing the microbial load. Generally, the protein solution is heatedto a temperature of about 70° to about 100° C., preferably about 85° toabout 95° C., for about 10 seconds to about 60 minutes, preferably about30 seconds to about 5 minutes. The heat treated acidified soy proteinsolution then may be cooled to a temperature of about 2° C. to about 60°C., preferably about 20° to about 35° C. The resulting acidified, heattreated soy protein solution then is dried.

The concentrated and optionally diafiltered protein solution may beraised in ionic strength by salt addition, if desired, to promote theformation of protein micellar mass upon dilution as an alternative tothe ionic strength adjustment operation described above.

Depending on the temperature employed in the concentration step andoptional diafiltration step and whether or not a pasteurization step iseffected, the concentrated protein solution may be warmed to atemperature of at least about 20° C., and up to about 60° C., preferablyabout 25° C. to about 40° C., to decrease the viscosity of theconcentrated protein solution to facilitate performance of thesubsequent dilution step and micelle formation. The concentrated proteinsolution should not be heated beyond a temperature above which micelleformation does not occur on dilution by chilled water.

The concentrated protein solution resulting from the concentration step,optional diafiltration step, optional ionic strength adjustment step,optional defatting step, optional adsorbent treatment step and optionalpasteurization step, then is diluted to effect micelle formation bymixing the concentrated protein solution with chilled water having thevolume required to achieve the degree of dilution desired. Depending onthe proportion of soy protein desired to be obtained by the micelleroute and the proportion from the supernatant, the degree of dilution ofthe concentrated protein solution may be varied. With lower dilutionlevels, in general, a greater proportion of the soy protein remains inthe aqueous phase.

When it is desired to provide the greatest proportion of the protein bythe micelle route, the concentrated protein solution is diluted by about5 fold to about 25 fold, preferably by about 10 fold to about 20 fold.

The chilled water with which the concentrated protein solution is mixedhas a temperature of less than about 15° C., generally about 1° to about15° C., preferably less than about 10° C., since improved yields ofprotein isolate in the form of protein micellar mass are attained withthese colder temperatures at the dilution factors used.

In a batch operation, the batch of concentrated protein solution isadded to a static body of chilled water having the desired volume, asdiscussed above. The dilution of the concentrated protein solution andconsequential decrease in ionic strength causes the formation of acloud-like mass of highly associated protein molecules in the form ofdiscrete protein droplets in micellar form. In the batch procedure, theprotein micelles are allowed to settle in the body of chilled water toform an aggregated, coalesced, dense, amorphous sticky gluten-likeprotein micellar mass (PMM). The settling may be assisted, such as bycentrifugation. Such induced settling decreases the liquid content ofthe protein micellar mass, thereby decreasing the moisture contentgenerally from about 70% by weight to about 95% by weight to a value ofgenerally about 50% by weight to about 80% by weight of the totalmicellar mass. Decreasing the moisture content of the micellar mass inthis way also decreases the occluded salt content of the micellar mass,and hence the salt content of the dried protein product.

Alternatively, the dilution operation may be carried out continuously bycontinuously passing the concentrated protein solution to one inlet of aT-shaped pipe, while the diluting water is fed to the other inlet of theT-shaped pipe, permitting mixing in the pipe. The diluting water is fedinto the T-shaped pipe at a rate sufficient to achieve the desireddegree of dilution of the concentrated protein solution.

The mixing of the concentrated protein solution and the diluting waterin the pipe initiates the formation of protein micelles and the mixtureis continuously fed from the outlet of the T-shaped pipe into a settlingvessel, from which, when full, supernatant is permitted to overflow. Themixture preferably is fed into the body of liquid in the settling vesselin a manner which minimizes turbulence within the body of liquid.

In the continuous procedure, the protein micelles are allowed to settlein the settling vessel to form an aggregated, coalesced, dense,amorphous, sticky, gluten-like protein micellar mass (PMM) and theprocedure is continued until a desired quantity of the PMM hasaccumulated in the bottom of the settling vessel, whereupon theaccumulated PMM is removed from the settling vessel. In lieu of settlingby sedimentation, the PMM may be separated continuously bycentrifugation.

By the utilization of a continuous process for the recovery of soyprotein micellar mass as compared to the batch process, the initialprotein extraction step can be significantly reduced in time for thesame level of protein extraction and significantly higher temperaturescan be employed in the extraction step. In addition, in a continuousoperation, there is less chance of contamination than in a batchprocedure, leading to higher product quality and the process can becarried out in more compact equipment.

The settled micellar mass is separated from the residual aqueous phaseor supernatant, such as by decantation of the residual aqueous phasefrom the settled mass or by centrifugation. The PMM may be used in thewet form or may be dried, by any convenient technique, such as spraydrying or freeze drying, to a dry form. The dry PMM has a high proteincontent, in excess of about 90 wt % protein, preferably at least about100 wt % protein (calculated as N×6.25) d.b., and is substantiallyundenatured. Alternatively, the wet PMM may be adjusted in pH to a pH ofabout 2.0 to about 4.0, preferably about 2.9 to about 3.2. The pHadjustment may be effected in any convenient manner, such as by additionof hydrochloric acid or phosphoric acid. The resulting acidified soyprotein solution then is dried. As a further alternative, the pHadjusted soy protein solution may be subjected to a heat treatment toinactivate heat labile anti-nutritional factors, such as the trypsininhibitors mentioned above. Such a heating step also provides theadditional benefit of reducing the microbial load. Generally, theprotein solution is heated to a temperature of about 70° to about 100°C., preferably about 85° to about 95° C., for about 10 seconds to about60 minutes, preferably about 30 seconds to about 5 minutes. The heattreated acidified soy protein solution then may be cooled to atemperature of about 2° C. to about 60° C., preferably about 20° toabout 35° C. The resulting acidified, heat treated soy protein solutionthen is dried.

In one aspect of the present invention, a calcium salt or other divalentsalt, preferably calcium chloride is added to the supernatant, which mayfirst be concentrated or partially concentrated in the manner describedbelow, to provide a conductivity of about 2 mS to about 30 mS,preferably 8 mS to about 15 mS. The calcium chloride added to thesupernatant may be in any desired form, such as a concentrated aqueoussolution thereof.

The addition of the calcium chloride has the effect of depositing phyticacid from the supernatant in the form of calcium phytate. The depositedphytate is recovered from the supernatant, such as by centrifugationand/or filtration to leave a clear solution.

The pH of the clear solution then may be adjusted to a value of about1.5 to about 4.4, preferably about 2.0 to about 4.0. The pH adjustmentmay be effected in any convenient manner, such as by the addition ofhydrochloric acid or phosphoric acid. If desired, the acidification stepmay be omitted from the various options described herein (other than theheat treatment mentioned below), once the precipitated phytate materialhas been removed.

The pH adjusted clear acidified aqueous soy protein solution may besubjected to a heat treatment to inactivate heat labile anti-nutritionalfactors, such as the trypsin inhibitors mentioned above. Such a heatingstep also provides the additional benefit of reducing the microbialload. Generally, the protein solution is heated to a temperature ofabout 70° to about 100° C., preferably about 85° to about 95° C., forabout 10 seconds to about 60 minutes, preferably about 30 seconds toabout 5 minutes. The heat treated acidified soy protein solution thenmay be cooled for further processing as described below, to atemperature of about 2° C. to about 60° C., preferably about 20° toabout 35° C.

The optionally pH-adjusted and optionally heat treated clear solution,if not already concentrated, is concentrated to increase the proteinconcentration thereof. Such concentration is effected using anyconvenient selective membrane technique, such as ultrafiltration ordiafiltration, using membranes with a suitable molecular weight cut-offpermitting low molecular weight species, including salt, carbohydrates,pigments, trypsin inhibitors and other low molecular weight materialsextracted from the protein source material, to pass through themembrane, while retaining a significant proportion of the soy protein inthe solution. Ultrafiltration membranes having a molecular weightcut-off of about 3,000 to 1,000,000 Daltons, preferably about 5,000 toabout 100,000 Daltons, having regard to differing membrane materials andconfiguration, may be used. Concentration of the protein solution inthis way also reduces the volume of liquid required to be dried torecover the protein. The protein solution generally is concentrated to aprotein concentration of about 50 g/L to about 400 g/L, preferably about100 to about 250 g/L, prior to drying. Such concentration operation maybe carried out in a batch mode or in a continuous operation, asdescribed above.

Where the supernatant is partially concentrated prior to the addition ofthe calcium salt and fully concentrated after removal of theprecipitate, the supernatant is first concentrated to a proteinconcentration of about 50 g/L or less, and, after removal of theprecipitate, then is concentrated to a protein concentration of about 50to about 400 g/L, preferably about 100 to about 250 g/L.

The protein solution may be subjected to a diafiltration step, before orafter partial or complete concentration, preferably using water or adilute saline solution. The diafiltration solution may be at its naturalpH, a pH equal to that of the protein solution being diafiltered or anypH in between. Such diafiltration may be effected using from about 2 toabout 40 volumes of diafiltration solution, preferably about 5 to about25 volumes of diafiltration solution. In the diafiltration operation,further quantities of contaminants are removed from the aqueous solutionby passage through the membrane with the permeate. The diafiltrationoperation may be effected until no significant further quantities ofcontaminants or visible colour are present in the permeate or until theprotein solution has been sufficiently purified. Such diafiltration maybe effected using the same membrane as for the concentration step.However, if desired, the diafiltration may be effected using a separatemembrane, such as a membrane having a molecular weight cut-off in therange of about 3,000 to about 1,000,000 daltons, preferably about 5,000to about 100,000 daltons, having regard to different membrane materialsand configuration.

The concentration step and the diafiltration step may be effected hereinin such a manner that the soy protein product subsequently recovered bydrying the concentrated and diafiltered retentate contains less thanabout 90 wt % protein (N×6.25) d.b., such as at least about 60 wt %protein (N×6.25) d.b. By partially concentrating and/or partiallydiafiltering the aqueous soy protein solution, it is possible to onlypartially remove contaminants. This protein solution may then be driedto provide a soy protein product with lower levels of purity. The soyprotein product is still able to produce clear protein solutions underacidic conditions.

An antioxidant may be present in the diafiltration medium during atleast part of the diafiltration step. The antioxidant may be anyconvenient antioxidant, such as sodium sulfite or ascorbic acid. Thequantity of antioxidant employed in the diafiltration medium depends onthe materials employed and may vary from about 0.01 to about 1 wt %,preferably about 0.05 wt %. The antioxidant serves to inhibit theoxidation of any phenolics present in the concentrated soy proteinisolate solution.

The concentration step and the diafiltration step may be effected at anyconvenient temperature, generally about 2° to about 60° C., preferablyabout 20° to about 35° C., and for the period of time to effect thedesired degree of concentration and diafiltration. The temperature andother conditions used to some degree depend upon the membrane equipmentused to effect the membrane processing, the desired proteinconcentration of the solution and the efficiency of the removal ofcontaminants to the permeate.

As mentioned above, the level of trypsin inhibitor activity in the finalsoy protein product can be controlled by manipulation of various processvariables.

As previously noted, heat treatment of the acidified aqueous soy proteinsolution may be used to inactivate heat-labile trypsin inhibitors. Thepartially concentrated or fully concentrated acidified soy proteinsolution may also be heat treated to inactivate heat labile trypsininhibitors.

In addition, the concentration and/or diafiltration steps may beoperated in a manner favorable for removal of trypsin inhibitors in thepermeate along with the other contaminants. Removal of the trypsininhibitors is promoted by using a membrane of larger pore size, such asabout 30,000 to 1,000,000 Da, operating the membrane at elevatedtemperatures, such as about 30 to about 60° C. and employing greatervolumes of diafiltration medium, such as about 20 to about 40 volumes.

Acidifying and membrane processing the diluted protein solution at alower pH, such as about 1.5 to about 3 may reduce the trypsin inhibitoractivity relative to processing the solution at a higher pH, such asabout 3 to about 4.4. When the protein solution is concentrated anddiafiltered at the low end of the pH range, it may be desired to raisethe pH of the retentate prior to drying. The pH of the concentrated anddiafiltered protein solution may be raised to the desired value, forexample pH 3, by the addition of any convenient food grade alkali suchas sodium hydroxide.

Further, a reduction in trypsin inhibitor activity may be achieved byexposing soy materials to reducing agents that disrupt or rearrange thedisulfide bonds of the inhibitors. Suitable reducing agents includesodium sulfite, cysteine and N-acetylcysteine.

The addition of such reducing agents may be effected at various stagesof the overall process. The reducing agent may be added with the soyprotein source material in the extraction step, may be added to theclarified aqueous soy protein solution following removal of residual soyprotein source material, may be added to the diafiltered retentatebefore dilution, may be added to the supernatant, may be added to theconcentrated and diafiltered calcium modified supernatant before dryingor may be dry blended with the dried soy protein product. The additionof the reducing agent may be combined with a heat treatment step and themembrane processing steps, as described above.

If it is desired to retain active trypsin inhibitors in the concentratedprotein solution, this can be achieved by eliminating or reducing theintensity of the heat treatment step, not utilizing reducing agents,operating the concentration and diafiltration steps at the higher end ofthe pH range, such as about 3 to about 4.4, utilizing a concentrationand diafiltration membrane with a smaller pore size, operating themembrane at lower temperatures and employing fewer volumes ofdiafiltration medium.

The concentrated and diafiltered aqueous protein solution may be treatedwith an adsorbent, such as powdered activated carbon or granulatedactivated carbon, to remove colour and/or odour compounds. Suchadsorbent treatment may be carried out under any convenient conditions,generally at the ambient temperature of the concentrated proteinsolution. For powdered activated carbon, an amount of about 0.025% toabout 5% w/v, preferably about 0.05% to about 2% w/v, is employed. Theadsorbent may be removed from the soy protein solution by any convenientmeans, such as by filtration.

The pH of the concentrated and optionally diafiltered and optionallyadsorbent treated protein solution may be adjusted to about 2.0 to about4.0, if a pH adjustment step has not already been employed. The pHadjusted, concentrated and optionally diafiltered and optionallyadsorbent treated protein solution may also be heat treated to reducethe level of trypsin inhibitor activity as described above.

The concentrated and optionally diafiltered and optionally adsorbenttreated protein solution is dried by any convenient technique, such asspray drying or freeze drying, to a dry form. The dried soy proteinproduct has a protein content of at least about 60 wt % (N×6.25) d.b.,preferably in excess of about 90 wt % (N×6.25) d.b., more preferably atleast about 100 wt %. The soy protein product is low in phytic acidcontent, generally less than about 1.5% by weight.

In one embodiment of the present invention, the supernatant from theformation of PMM may be processed directly to form a soy protein productutilizing the steps described above while omitting the addition ofcalcium chloride. The soy protein product so formed has a proteincontent of at least about 60 wt % (N×6.25) d.b., preferably in excess ofabout 90 wt % (N×6.25) d.b., more preferably at least about 100 wt %.

The soy protein products produced herein are soluble in an acidicaqueous environment, making the products ideal for incorporation intobeverages, both carbonated and uncarbonated, to provide proteinfortification thereto. Such beverages have a wide range of acidic pHvalues, ranging from about 2.5 to about 5. The soy protein productsprovided herein may be added to such beverages in any convenientquantity to provide protein fortification to such beverages, forexample, to provide at least about 5 g of soy protein per serving. Theadded soy protein product dissolves in the beverage and does not impairthe clarity of the beverage, even after thermal processing. The soyprotein product may be blended with dried beverage prior toreconstitution of the beverage by dissolution in water. In some case,modification of the normal formulation of the beverage to tolerate thecomposition of the invention may be necessary where components presentin the beverage may adversely affect the ability of the composition toremain dissolved in the beverage.

EXAMPLES Example 1

This Example illustrates the production of protein micellar mass (S300),supernatant derived protein isolate (S200) and calcium modifiedsupernatant derived protein isolate (S200Ca) from soy.

‘a’ kg of defatted, minimally heat processed soy flour was added to ‘b’L of ‘c’ M NaCl solution at ambient temperature and agitated for 60minutes to provide an aqueous protein solution. The residual soy flourwas removed and the resulting protein solution was clarified bycentrifugation and filtration to produce ‘d’ L of filtered proteinsolution having a protein content of ‘e’% by weight.

The protein extract solution was reduced to ‘f’ kg by concentration on a‘g’ membrane having a molecular weight cutoff of ‘h’ Daltons producing aconcentrated protein solution with a protein content of ‘i’% by weight.

The conductivity of the concentrated protein solution was ‘j’ mS.Concentrated sodium chloride solution was added to the retentate toraise the conductivity to'k′ mS. The concentrated protein solution at‘1’° C. was then diluted ‘m’ into cold RO water having a temperature‘n’° C. A white cloud formed immediately. The supernatant was removedand the precipitated, viscous, sticky mass (PMM) was recovered bycentrifugation in a yield of ‘o’ wt % of the filtered protein solution.The dried PMM derived protein was found to have a protein content of‘p’% (N×6.25) d.b. The product was given a designation ‘q’ S300.

The parameters ‘a’ to ‘q’ are set forth in the following Table 1:

TABLE 1 Parameters for the production of S300 q S005-J27-08AS005-K19-08A a 10 10 b 200 200 c 0.15 0.50 d 185 165 e 0.70 1.34 f 5.2812.06 g PES PES h 100,000 100,000 i 21.28 17.51 j 9.45 24.9 k 21.4 24.9l 27.8 30 m 1:10 1:5 n 1.6 4 o 18.5 20.8 p 91.31 99.66

The supernatants from these two runs were processed in different ways.The supernatant from the S005-J27-08A run was processed without calciummodification. In this run, 65 L of supernatant was concentrated to avolume of 5 L on a PES membrane with a molecular weight cutoff of 10,000Daltons then diafiltered with 25 L of reverse osmosis purified water onthe same membrane. The diafiltered retentate had a protein concentrationof 12.60 wt %. With the additional protein recovered from thesupernatant, the overall recovery of the filtered protein solution was69.2%. The diafiltered retentate was dried to form a product with aprotein content of 98.76% (N×6.25) d.b. The product was given thedesignation S005-J27-08A S200.

The supernatant from run S005-K19-08A was processed with calciummodification. To 65 L of supernatant was added 0.336 kg of CaCl₂, whichraised the conductivity of the solution from 6.31 mS to 12.65 mS. Theprecipitate that formed was removed by centrifugation and then the pH ofthe centrate adjusted to 3 with diluted HCI. The acidified centrate wasthen concentrated from a volume of 66 L to a volume of 5 L on a PESmembrane with a molecular weight cut-off of 10,000 Daltons. Theconcentrate was then diafiltered on the same membrane with 25 L ofreverse osmosis purified water adjusted to pH 3 with diluted HCl. Withthe additional protein recovered from the supernatant, the overallrecovery of the filtered protein solution was 37.1%. The diafilteredretentate was dried to produce a product with a protein content of98.01% (N×6.25) d.b. The product was given the designation S005-K19-08AS200Ca.

The colour of the dry powdered products was assessed with a HunterLabColorQuest XE instrument in reflectance mode. The colour values are setforth in the following Table 2:

TABLE 2 HunterLab scores for dry products sample L* a* b* S005-J27-08AS300 87.06 −0.28 10.04 S005-K19-08A S300 85.98 0.72 10.91 S005-J27-08AS200 84.51 0.56 10.51 S005-K19-08A S200Ca 86.87 0.58 9.53

As may be seen from Table 2, the dry colour of all the products wasquite light.

Example 2

This Example contains an evaluation of the heat stability in water ofthe soy protein isolates produced by the method of Example 1 (S300,S200, S200Ca).

A 2% w/v protein solution of each product in water was produced and thepH adjusted to 3. The clarity of these solutions was assessed by hazemeasurement with the HunterLab ColorQuest XE instrument in transmissionmode. The solutions were then heated to 95° C., held at this temperaturefor 30 seconds and then immediately cooled to room temperature in an icebath. The clarity of the heat treated solutions was then measured again.

The clarity of the protein solutions before and after heating is setforth in the following Table 3:

TABLE 3 Effect of heat treatment on clarity of various samples Haze (%)Haze (%) sample before heating after heating S005-J27-08A S300 24.9 21.1S005-K19-08A S300 30.5 29.6 S005-J27-08A S200 11.0 3.2 S005-K19-08AS200Ca 7.3 7.9

As can be seen in Table 3, the S200 and S200Ca samples gave quite clearsolutions in water at pH 3. The solutions of the S300 samples were notas clear. All of the samples were heat stable, with the haze levelessentially staying constant upon heating, or actually improving.

Example 3

This Example contains an evaluation of the solubility in water of thesoy protein isolates produced by the method of Example 1 (S300, S200,S200Ca). Solubility was tested based on protein solubility (termedprotein method, a modified version of the procedure of Morr et al., J.Food Sci. 50:1715-1718) and total product solubility (termed pelletmethod).

Sufficient protein powder to supply 0.5 g of protein was weighed into abeaker and then a small amount of reverse osmosis (RO) purified waterwas added and the mixture stirred until a smooth paste formed.Additional water was then added to bring the volume to approximately 45ml. The contents of the beaker were then slowly stirred for 60 minutesusing a magnetic stirrer. The pH was determined immediately afterdispersing the protein and was adjusted to the appropriate level (2, 3,4, 5, 6 or 7) with diluted NaOH or HCl. A sample was also prepared atnatural pH. For the pH adjusted samples, the pH was measured andcorrected two times during the 60 minutes stirring. After the 60 minutesof stirring, the samples were made up to 50 ml total volume with ROwater, yielding a 1% w/v protein dispersion. The protein content of thedispersions was measured using a LECO FP528 Nitrogen Determinator.Aliquots (20 ml) of the dispersions were then transferred to pre-weighedcentrifuge tubes that had been dried overnight in a 100° C. oven thencooled in a desiccator and the tubes capped. The samples werecentrifuged at 7800 g for 10 minutes, which sedimented insolublematerial and yielded a clear supernatant. The protein content of thesupernatant was measured by LECO analysis and then the supernatant andthe tube lids were discarded and the pellet material dried overnight inan oven set at 100° C. The next morning the tubes were transferred to adesiccator and allowed to cool. The weight of dry pellet material wasrecorded. The dry weight of the initial protein powder was calculated bymultiplying the weight of powder used by a factor of ((100−moisturecontent of the powder (%))/100). Solubility of the product was thencalculated two different ways:

Solubility(protein method)(%)=(% protein in supernatant% protein ininitial dispersion)×100  1)

Solubility(pellet method)(%)=(1−(weight dry insoluble pelletmaterial/((weight of 20 ml of dispersion/weight of 50 ml ofdispersion)×initial weight dry protein powder)))×100  2)

The natural pH values of the protein isolates produced in Example 1 inwater (1% protein) are shown in Table 4:

TABLE 4 Natural pH of protein solution prepared in water at 1% proteinBatch Product Natural pH S005-J27-08A S300 6.67 S005-K19-08A S300 6.76S005-J27-08A S200 6.70 S005-K19-08A S200Ca 3.29

The solubility results obtained are set forth in the following Tables 5and 6:

TABLE 5 Solubility of products at different pH values based on proteinmethod Solubility (Protein method) (%) pH pH pH pH pH pH Nat. BatchProduct 2 3 4 5 6 7 pH S005-J27- S300 100 94.2 43.4 19.1 91.9 99.1 95.008A S005-K19- S300 100 100 85.3 8.1 23.7 100 94.7 08A S005-J27- S20091.5 100 98.8 0.0 76.7 94.4 89.5 08A S005-K19- S200Ca 94.7 100 100 20 3866.3 100 08A

TABLE 6 Solubility of products at different pH values based on pelletmethod Solubility (pellet method) (%) pH pH pH pH pH pH Nat. BatchProduct 2 3 4 5 6 7 pH S005-J27- S300 97.1 97.0 55.4 29.3 91.7 94.5 86.908A S005-K19- S300 96.5 96.1 76.3 5.7 29.1 93.1 86.8 08A S005-J27- S20096.9 97.8 96.3 15.1 86.1 97.9 98.1 08A S005-K19- S200Ca 98.2 95.8 97.231.4 55.0 71.1 98.3 08A

As can be seen from the results of Tables 5 and 6, the S300 productswere very soluble at pH values 2, 3 and 7. The S200 was very soluble atpH 2 to 4 and 7. The S200Ca was very soluble in the range of pH 2 to 4.

Example 4

This Example contains an evaluation of the clarity in water of the soyprotein isolates produced by the method of Example 1 (S300, S200,S200Ca).

The clarity of the 1% w/v protein solutions prepared as described inExample 3 was assessed by measuring the absorbance at 600 nm, with alower absorbance score indicating greater clarity. Analysis of thesamples on a HunterLab ColorQuest XE instrument in transmission modealso provided a percentage haze reading, another measure of clarity.

The clarity results are set forth in the following Tables 7 and 8:

TABLE 7 Clarity of protein solutions at different pH values as assessedby A600 A600 pH pH pH pH pH pH Nat. Batch Product 2 3 4 5 6 7 pHS005-J27- S300 0.025 0.064 >3.0 >3.0 1.568 0.819 2.482 08A S005-K19-S300 0.059 0.117 1.995 >3.0 >3.0 0.319 0.468 08A S005-J27- S200 0.0530.066 0.127 >3.0 1.064 0.070 0.080 08A S005-K19- S200Ca 0.031 0.0400.066 >3.0 >3.0 1.922 0.047 08A

TABLE 8 Clarity of protein solutions at different pH values as assessedby HunterLab analysis HunterLab haze reading (%) pH pH pH pH pH pH Nat.Batch Product 2 3 4 5 6 7 pH S005-J27- S300 8.1 16.3 98.9 99.9 97.6 89.598.8 08A S005-K19- S300 5.8 16.9 92.4 93.4 93.4 40.2 54.1 08A S005-J27-S200 5.6 6.4 14.4 97.4 86.5 8.1 9.2 08A S005-K19- S200Ca 1.2 3.3 7.193.6 92.9 92.4 2.9 08A

As can be seen from the results of Tables 7 and 8, solutions of S300were clear at pH 2 and slightly hazy at pH 3. Solutions of this productat the higher pH values were quite hazy. Solutions of S200 and S200Cawere clear in the pH range 2 to 4 and the S200 solution was also clearat natural pH and pH 7.

Example 5

This Example contains an evaluation of the solubility in a soft drink(Sprite) and sports drink (Orange Gatorade) of the soy protein isolatesproduced by the method of Example 1 (S300, S200, S200Ca). The solubilitywas determined with the protein added to the beverages with no pHcorrection and again with the pH of the protein fortified beveragesadjusted to the level of the original beverages.

When the solubility was assessed with no pH correction, a sufficientamount of protein powder to supply 1 g of protein was weighed into abeaker and a small amount of beverage was added and stirred until asmooth paste formed. Additional beverage was added to bring the volumeto 50 ml, and then the solutions were stirred slowly on a magneticstirrer for 60 minutes to yield a 2% protein w/v dispersion. The proteincontent of the samples was analyzed using a LECO FP528 NitrogenDeterminator then an aliquot of the protein containing beverages wascentrifuged at 7800 g for 10 minutes and the protein content of thesupernatant measured.

Solubility(%)=(% protein in supernatant% protein in initialdispersion)×100

When the solubility was assessed with pH correction, the pH of the softdrink (Sprite) (3.39) and sports drink (Orange Gatorade) (3.19) withoutprotein was measured. A sufficient amount of protein powder to supply 1g of protein was weighed into a beaker and a small amount of beveragewas added and stirred until a smooth paste formed. Additional beveragewas added to bring the volume to approximately 45 ml, and then thesolutions were stirred slowly on a magnetic stirrer for 60 minutes. ThepH of the protein containing beverages was measured and then adjusted tothe original no-protein pH with HCl or NaOH as necessary. The totalvolume of each solution was then brought to 50 ml with additionalbeverage, yielding a 2% protein w/v dispersion. The protein content ofthe samples was analyzed using a LECO FP528 Nitrogen Determinator thenan aliquot of the protein containing beverages was centrifuged at 7800 gfor 10 minutes and the protein content of the supernatant measured.

Solubility(%)=(% protein in supernatant% protein in initialdispersion)×100

The results obtained are set forth in the following Table 9:

TABLE 9 Solubility of products in Sprite and Orange Gatorade no pHcorrection pH correction Solubility Solubility Solubility (%) inSolubility (%) in (%) in Orange (%) in Orange Batch Product SpriteGatorade Sprite Gatorade S005-J27-08A S300 25.6 42.2 87.9 90.3S005-K19-08A S300 4.8 71.0 95.3 85.2 S005-J27-08A S200 17.3 69.9 66.574.4 S005-K19-08A S200Ca 95.7 100 94.1 100

As can be seen from the results of Table 9, the S200Ca was the productwith the best solubility in the Sprite and Orange Gatorade. This is anacidified product and so had little effect on the beverage pH. Theremaining products were not acidified and so their solubility wasimproved by pH correction of the beverages. After pH correction, thesolubility of the S300 products was quite good but the solubility of theS200 was surprisingly low, given the solubility results obtained inwater in Example 3.

Example 6

This Example contains an evaluation of the clarity in a soft drink andsports drink of the soy protein isolates produced by the method ofExample 1 (S300, S200, S200Ca).

The clarity of the 2% w/v protein dispersions prepared in soft drink(Sprite) and sports drink (Orange Gatorade) in Example 5 were assessedusing the methods described in Example 4. For the absorbancemeasurements at 600 nm, the spectrophotometer was blanked with theappropriate beverage before the measurement was performed.

The results obtained are set forth in the following Tables 10 and 11:

TABLE 10 Clarity (A600) of products in Sprite and Orange Gatorade no pHcorrection pH correction A600 in A600 in A600 in Orange A600 in OrangeBatch Product Sprite Gatorade Sprite Gatorade S005-J27-08AS300 >3.0 >3.0 1.730 1.740 S005-K19-08A S300 >3.0 >3.0 1.339 1.028S005-J27-08A S200 >3.0 2.816 1.560 1.560 S005-K19-08A S200Ca 0.084 0.0190.093 0.071

TABLE 11 HunterLab haze readings for products in Sprite and OrangeGatorade no pH correction pH correction haze haze haze (%) in haze (%)in (%) in Orange (%) in Orange Batch Product Sprite Gatorade SpriteGatorade no protein 0.0 44.0 0.0 44.0 S005-J27-08A S300 97.7 98.1 89.389.9 S005-K19-08A S300 93.6 93.5 94.9 86.3 S005-J27-08A S200 97.4 98.288.6 90.4 S005-K19-08A S200Ca 12.3 46.7 19.5 53.3

As can be seen from the results of Tables 10 and 11, the S200Ca producthad the least impact on clarity in Sprite and Orange Gatorade. However,the S200Ca in Sprite was slightly hazy, particularly when tested with pHcorrection. The Sprite and Orange Gatorade samples containing S300 andS200 were very hazy regardless of whether pH correction was employed.

Example 7

This Example illustrates the production of a soy protein isolate derivedfrom concentrated retentate (S500) from a sodium chloride extraction.

12.5 kg of defatted, minimally heat processed soy flour was added to 125L of 0.15 M NaCl solution at ambient temperature and agitated for 30minutes to provide an aqueous protein solution. The residual soy flourwas removed and the resulting protein solution was clarified bycentrifugation and filtration to produce 97 L of filtered proteinsolution having a protein content of 1.14% by weight.

The protein extract solution was reduced in volume to 7 L byconcentration on a PVDF membrane having a molecular weight cutoff of5,000 daltons, producing a concentrated protein solution with a proteincontent of 14.83% by weight.

The concentrated protein solution was then diafiltered using 14 L of0.075 M NaCl solution. The diafiltered retentate had a final weight of6.14 kg and a protein content of 14.16% by weight in a yield of 78.4 wt% of the filtered protein solution. The diafiltered retentate was driedto form a product with a protein content of 95.45% (N×6.25) d.b. Theproduct was given the designation S005-L17-08A S500.

A 3.2% w/v protein solution of S500 was prepared in water and the pHlowered to 3 with diluted HCl. The colour and clarity was then assessedusing a HunterLab ColorQuest XE instrument operated in transmissionmode.

The colour and clarity values are set forth in the following Table 12:

TABLE 12 HunterLab scores for 3.2% protein solution of S005-L17-08A S500at pH 3 sample L* a* b* haze (%) S500 94.86 −1.15 15.45 22.0

As may be seen from Table 12, the colour of the S500 solution at pH 3was quite light but the solution was also hazy.

The colour of the dry powder was also assessed with the HunterLabColorQuest XE instrument in reflectance mode. The colour values are setforth in the following Table 13:

TABLE 13 HunterLab scores for dry S005-L17-08A S500 sample L* a* b* S50084.71 0.14 14.88

As may be seen from Table 13, the dry colour of the product was quitelight.

Example 8

This Example contains an evaluation of the heat stability in water ofthe soy protein isolate produced by the method of Example 7 (S500).

A 2% w/v protein solution of the product in water was produced and thepH adjusted to 3. The clarity of this solution was assessed by hazemeasurement with a HunterLab ColorQuest XE instrument in transmissionmode. The solution was then heated to 95° C., held at this temperaturefor 30 seconds and then immediately cooled to room temperature in an icebath. The clarity of the heat treated solution was then measured again.

The clarity of the protein solution before and after heating is setforth in the following Table 14:

TABLE 14 Effect of heat treatment on clarity of S005-L17-08A S500solution Haze (%) Haze (%) sample before heating after heating S500 7.99.8

As can be seen in Table 14, the S500 sample gave quite a clear solutionin water at pH 3. The sample was heat stable, with the haze level onlyslightly changed upon heating.

Example 9

This Example contains an evaluation of the solubility in water of thesoy protein isolate produced by the method of Example 7 (S500).Solubility was tested based on protein solubility (termed proteinmethod, a modified version of the procedure of Mon et al., J. Food Sci.50:1715-1718) and total product solubility (termed pellet method).

Sufficient protein powder to supply 0.5 g of protein was weighed into abeaker and then a small amount of reverse osmosis (RO) purified waterwas added and the mixture stirred until a smooth paste formed.Additional water was then added to bring the volume to approximately 45ml. The contents of the beaker were then slowly stirred for 60 minutesusing a magnetic stirrer. The pH was determined immediately afterdispersing the protein and was adjusted to the appropriate level (2, 3,4, 5, 6 or 7) with diluted NaOH or HCl. A sample was also prepared atnatural pH. For the pH adjusted samples, the pH was measured andcorrected two times during the 60 minutes stirring. After the 60 minutesof stirring, the samples were made up to 50 ml total volume with ROwater, yielding a 1% w/v protein dispersion. The protein content of thedispersions was measured using a LECO FP528 Nitrogen Determinator.Aliquots (20 ml) of the dispersions were then transferred to pre-weighedcentrifuge tubes that had been dried overnight in a 100° C. oven thencooled in a desiccator and the tubes capped. The samples werecentrifuged at 7800 g for 10 minutes, which sedimented insolublematerial and yielded a clear supernatant. The protein content of thesupernatant was measured by LECO analysis and then the supernatant andthe tube lids were discarded and the pellet material dried overnight inan oven set at 100° C. The next morning the tubes were transferred to adesiccator and allowed to cool. The weight of dry pellet material wasrecorded. The dry weight of the initial protein powder was calculated bymultiplying the weight of powder used by a factor of ((100−moisturecontent of the powder (%))/100). Solubility of the product was thencalculated two different ways:

Solubility(protein method)(%)=(% protein in supernatant% protein ininitial dispersion)×100  1)

Solubility(pellet method)(%)=(1−(weight dry insoluble pelletmaterial/((weight of 20 ml of dispersion/weight of 50 ml ofdispersion)×initial weight dry protein powder)))×100  2)

The natural pH value of the protein isolate produced in Example 7 inwater (1% protein) is shown in Table 15:

TABLE 15 Natural pH of S500 solution prepared in water at 1% proteinBatch Product Natural pH S005-L17-08A S500 6.61

The solubility results obtained are set forth in the following Tables 16and 17:

TABLE 16 Solubility of S500 at different pH values based on proteinmethod Solubility (protein method) (%) pH pH pH pH pH pH Nat. BatchProduct 2 3 4 5 6 7 pH S005-L17- S500 92.6 100 60.4 26.9 88.3 100 92.608A

TABLE 17 Solubility of S500 at different pH values based on pelletmethod Solubility (pellet method) (%) pH pH pH pH pH pH Nat. BatchProduct 2 3 4 5 6 7 pH S005-L17- S500 97.8 97.5 68.3 30.3 84.9 97.4 97.608A

As can be seen from the results of Tables 16 and 17, the S500 productwas very soluble at pH 2, 3 and 7 and at the natural pH.

Example 10

This Example contains an evaluation of the clarity in water of the soyprotein isolate produced by the method of Example 7 (S500).

The clarity of the 1% w/v protein solution prepared as described inExample 9 was assessed by measuring the absorbance at 600 nm, with alower absorbance score indicating greater clarity. Analysis of thesamples on a HunterLab ColorQuest XE instrument in transmission modealso provided a percentage haze reading, another measure of clarity.

The clarity results are set forth in the following Tables 18 and 19:

TABLE 18 Clarity of S500 solution at different pH values as assessed byA600 A600 pH pH pH pH pH pH Nat. Batch Product 2 3 4 5 6 7 pH S005-L17-S500 0.020 0.044 >3.0 >3.0 1.499 0.048 0.061 08A

TABLE 19 Clarity of S500 solution at different pH values as assessed byHunterLab analysis HunterLab haze reading (%) pH pH pH pH pH pH Nat.Batch Product 2 3 4 5 6 7 pH S005-L17- S500 0.6 6.5 95.3 95.9 90.8 7.05.5 08A

As can be seen from the results of Tables 18 and 19, solutions of S500had excellent clarity at pH 2, 3 and 7 and at natural pH.

Example 11

This Example contains an evaluation of the solubility in a soft drink(Sprite) and sports drink (Orange Gatorade) of the soy protein isolateproduced by the method of Example 7 (S500). The solubility wasdetermined with the protein added to the beverages with no pH correctionand again with the pH of the protein fortified beverages adjusted to thelevel of the original beverages.

When the solubility was assessed with no pH correction, a sufficientamount of protein powder to supply 1 g of protein was weighed into abeaker and a small amount of beverage was added and stirred until asmooth paste formed. Additional beverage was added to bring the volumeto 50 ml, and then the solutions were stirred slowly on a magneticstirrer for 60 minutes to yield a 2% protein w/v dispersion. The proteincontent of the samples was analyzed using a LECO FP528 NitrogenDeterminator then an aliquot of the protein containing beverages wascentrifuged at 7800 g for 10 minutes and the protein content of thesupernatant measured.

Solubility(%)=(% protein in supernatant% protein in initialdispersion)×100

When the solubility was assessed with pH correction, the pH of the softdrink (Sprite) (3.39) and sports drink (Orange Gatorade) (3.19) withoutprotein was measured. A sufficient amount of protein powder to supply 1g of protein was weighed into a beaker and a small amount of beveragewas added and stirred until a smooth paste formed. Additional beveragewas added to bring the volume to approximately 45 ml, and then thesolutions were stirred slowly on a magnetic stirrer for 60 minutes. ThepH of the protein containing beverages was measured and then adjusted tothe original no-protein pH with HCl or NaOH as necessary. The totalvolume of each solution was then brought to 50 ml with additionalbeverage, yielding a 2% protein w/v dispersion. The protein content ofthe samples was analyzed using a LECO FP 528 Nitrogen Determinator thenan aliquot of the protein containing beverages was centrifuged at 7800 gfor 10 minutes and the protein content of the supernatant measured.

Solubility(%)=(% protein in supernatant% protein in initialdispersion)×100

The results obtained are set forth in the following Table 20:

TABLE 20 Solubility of S500 in Sprite and Orange Gatorade no pHcorrection pH correction Solubility Solubility Solubility (%) inSolubility (%) in (%) in Orange (%) in Orange Batch Product SpriteGatorade Sprite Gatorade S005-L17-08A S500 22.5 50.0 82.0 79.9

As can be seen from the results of Table 20, the S500 was not verysoluble in the beverages without pH adjustment. This can partially beattributed to the fact that the S500 is not an acidified product.Correction of the pH did improve the solubility of S500 in bothbeverages, although the protein was still not completely soluble.

Example 12

This Example contains an evaluation of the clarity in a soft drink andsports drink of the soy protein isolate produced by the method ofExample 7 (S500).

The clarity of the 2% w/v protein dispersions prepared in soft drink(Sprite) and sports drink (Orange Gatorade) in Example 11 were assessedusing the methods described in Example 10. For the absorbancemeasurements at 600 nm, the spectrophotometer was blanked with theappropriate beverage before the measurement was performed.

The results obtained are set forth in the following Tables 21 and 22:

TABLE 21 Clarity (A600) of S500 in Sprite and Orange Gatorade no pHcorrection pH correction A600 in A600 in A600 in Orange A600 in OrangeBatch Product Sprite Gatorade Sprite Gatorade S005-L17-08AS500 >3.0 >3.0 1.056 1.710

TABLE 22 HunterLab haze readings for S500 in Sprite and Orange Gatoradeno pH correction pH correction haze haze haze (%) in haze (%) in (%) inOrange (%) in Orange Batch Product Sprite Gatorade Sprite Gatorade noprotein 0.0 44.0 0.0 44.0 S005-L17-08A S500 97.5 98.1 83.6 98.2

As may be seen from the results in Tables 21 and 22, Sprite and OrangeGatorade with added S500 were very hazy, with perhaps only slightimprovement achieved by correcting the pH.

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, there are produced soy protein isolateswhich can provide heat stable and clear aqueous solutions at acid pHvalues. Modifications are possible within the scope of this invention.

1. A process of preparing a soy protein product having a protein contentof at least about 60 wt % (N×6.25) on a dry weight basis, whichcomprises: adding calcium salt or other divalent salt to supernatantfrom the precipitation of a soy protein micellar mass to provide aconductivity of about 2 mS to about 30 mS, removing precipitate from theresulting solution to leave a clear solution, optionally adjusting thepH of the clear solution to about 1.5 to about 4.4, concentrating theoptionally pH-adjusted clear solution to a protein content of about 50to about 400 g/L to provide a clear concentrated soy protein solution,optionally diafiltering the clear concentrated protein solution, anddrying the concentrated solution.
 2. The process of claim 1 wherein saidcalcium salt is calcium chloride.
 3. The process of claim 1 wherein saidcalcium salt is added to the supernatant to provide a conductivity ofabout 8 to about 15 mS.
 4. The process of claim 1 wherein the optionallypH adjusted clear solution is concentrated to a protein concentration ofabout 100 to about 250 g/L.
 5. The process of claim 1 wherein theoptionally pH adjusted clear solution is concentrated using a membranehaving a molecular weight cut-off of about 3,000 to about 1,000,000Daltons.
 6. The process of claim 5 wherein the optionally pH adjustedclear solution is concentrated using a membrane having a molecularweight cut-off of about 5,000 to about 100,000 Daltons.
 7. The processof claim 1 wherein a diafiltration step is effected using water,acidified water, dilute salt solution or an acidified, dilute saltsolution on the soy protein solution before or after completeconcentration thereof.
 8. The process of claim 7 wherein saiddiafiltration step is effected using about 2 to about 40 volumes ofdiafiltration solution.
 9. The process of claim 8 wherein saiddiafiltration step is effected using about 5 to about 25 volumes ofdiafiltration solution.
 10. The process of claim 7 wherein saiddiafiltration step is effected using a membrane having a molecularweight cut-off of about 3,000 to about 1,000,000 Daltons.
 11. Theprocess of claim 10 wherein said diafiltration step is effected using amembrane having a molecular weight cut-off of about 5,000 to about100,000 Daltons.
 12. The process of claim 7 wherein an antioxidant ispresent during at least part of the diafiltration step.
 13. The processof claim 1 wherein the concentrated and optionally diafiltered soyprotein solution is treated with an adsorbent to remove colour and/orodour compounds prior to said drying step.
 14. The process of claim 1wherein said soy protein product has a protein content of about 60 toabout 90 wt % (N×6.25) d.b.
 15. The process of claim 1 wherein said soyprotein product is an isolate having a protein content of at least about90 wt % (N×6.25) d.b.
 16. The process of claim 1 wherein said soyprotein product is an isolate having a protein content of at least about100 wt % (N×6.25) d.b.
 17. The process of claim 1 wherein the pH of theclear solution is adjusted to about 2.0 to about 4.0.
 18. The process ofclaim 1 wherein the concentrated and optionally diafiltered soy proteinsolution, if not already acidified, is acidified to a pH of about 2.0 toabout 4.0 prior to drying.
 19. The process of claim 1 wherein said clearacidified soy protein solution is subjected to a heat treatment step toinactivate heat-labile anti-nutritional factors.
 20. The process ofclaim 19 wherein the anti-nutritional factors are heat-labile trypsininhibitors.
 21. The process of claim 19 wherein the heat treatment stepalso pasteurizes the acidified clear aqueous protein solution.
 22. Theprocess of claim 19 wherein said heat-treatment is effected at atemperature of about 70° to about 100° C. for about 10 seconds to aboutsixty minutes.
 23. The process of claim 22 wherein said heat-treatmentis effected at a temperature of about 85° to about 95° C. for about 30seconds to about 5 minutes.
 24. The process of claim 19 wherein theheat-treated clear acidified soy protein solution is cooled to atemperature of about 2° to about 60° C. for further processing.
 25. Theprocess of claim 24 wherein the heat-treated clear acidified soy proteinsolution is cooled to a temperature of about 20° to about 35° C. forfurther processing.
 26. The process of claim 7 wherein the concentrationand/or optional diafiltration step are operated in a manner favourableto the removal of trypsin inhibitors.
 27. The process of claim 1 whereina reducing agent is added to the supernatant to disrupt or rearrange thedisulfide bonds of trypsin inhibitors to achieve a reduction in trypsininhibitor activity.
 28. The process of claim 7 wherein a reducing agentis present during the concentration and/or optional diafiltration stepto disrupt or rearrange the disulfide bonds of trypsin inhibitors toachieve a reduction in trypsin inhibitor activity.
 29. The process ofclaim 1 wherein a reducing agent is added to the concentrated andoptionally diafiltered soy protein solution prior to drying and/or thedried soy protein product to disrupt or rearrange the disulfide bonds oftrypsin inhibitors to achieve a reduction in trypsin inhibitor activity.30. A process of preparing a soy protein product having a proteincontent of at least about 60 wt % (N×6.25) on a dry weight basis, whichcomprises: partially concentrating the supernatant from theprecipitation of a soy protein micellar mass to a protein concentrationof less than about 50 g/L, adding calcium salt or other divalent salt tothe partially concentrated supernatant to provide a conductivity ofabout 2 mS to about 30 mS, removing precipitate from the resultingsolution to leave a clear solution, optionally adjusting the pH of theclear solution to about 1.5 to about 4.4, further concentrating theoptionally pH-adjusted clear solution to a protein content of about 50to about 400 g/L to provide a clear concentrated soy protein solution,optionally diafiltering the clear concentrated protein solution, anddrying the concentrated solution.
 31. The process of claim 30 whereinsaid calcium salt is calcium chloride.
 32. The process of claim 30wherein said calcium salt is added to the partially concentratedsupernatant to provide a conductivity of about 8 to about 15 mS.
 33. Theprocess of claim 30 wherein said optionally pH adjusted clear solutionis further concentrated to a protein concentration of about 100 to about250 g/L.
 34. The process of claim 30 wherein said concentration stepsare effected using a membrane having a molecular weight cut-off of about3,000 to about 1,000,000 Daltons.
 35. The process of claim 34 whereinsaid concentration steps are effected using a membrane having amolecular weight cut-off of about 5,000 to about 100,000 Daltons. 36.The process of claim 30 wherein a diafiltration step is effected usingwater, acidified water, dilute salt solution or an acidified, dilutesalt solution on the soy protein solution before or after partial orcomplete concentration thereof.
 37. The process of claim 36 wherein saiddiafiltration step is effected using about 2 to about 40 volumes ofdiafiltration solution.
 38. The process of claim 37 wherein saiddiafiltration step is effected using about 5 to about 25 volumes ofdiafiltration solution.
 39. The process of claim 36 wherein saiddiafiltration step is effected using a membrane having a molecularweight cut-off of about 3,000 to about 1,000,000 Daltons.
 40. Theprocess of claim 39 wherein said diafiltration step is effected using amembrane having a molecular weight cut-off of about 5,000 to about100,000 Daltons.
 41. The process of claim 36 wherein an antioxidant ispresent during at least part of the diafiltration step.
 42. The processof claim 30 wherein the concentrated and optionally diafiltered soyprotein solution is treated with an adsorbent to remove colour and/orodour compounds prior to said drying step.
 43. The process of claim 30wherein said soy protein product has a protein content of about 60 toabout 90 wt % (N×6.25) d.b.
 44. The process of claim 30 wherein said soyprotein product is an isolate having a protein content of at least about90 wt % (N×6.25) d.b.
 45. The process of claim 30 wherein said soyprotein product is an isolate having a protein content of at least about100 wt % (N×6.25) d.b.
 46. The process of claim 30 wherein the pH of theclear solution is adjusted to about 2.0 to about 4.0.
 47. The process ofclaim 30 wherein the concentrated and optionally diafiltered soy proteinsolution, if not already acidified, is acidified to a pH of about 2.0 toabout 4.0 prior to drying.
 48. The process of claim 30 wherein saidclear acidified soy protein solution is subjected to a heat treatmentstep to inactivate heat-labile anti-nutritional factors.
 49. The processof claim 48 wherein the anti-nutritional factors are heat-labile trypsininhibitors.
 50. The process of claim 48 wherein the heat treatment stepalso pasteurizes the acidified clear aqueous protein solution.
 51. Theprocess of claim 48 wherein said heat-treatment is effected at atemperature of about 70° to about 100° C. for about 10 seconds to aboutsixty minutes.
 52. The process of claim 51 wherein said heat-treatmentis effected at a temperature of about 85° to about 95° C. for about 30seconds to about 5 minutes.
 53. The process of claim 48 wherein theheat-treated clear acidified soy protein solution is cooled to atemperature of about 2° to about 60° C. for further processing.
 54. Theprocess of claim 53 wherein the heat-treated clear acidified soy proteinsolution is cooled to a temperature of about 20° to about 35° C. forfurther processing.
 55. The process of claim 36 wherein theconcentration and/or optional diafiltration step are operated in amanner favourable to the removal of trypsin inhibitors.
 56. The processof claim 30 wherein a reducing agent is added to the supernatant todisrupt or rearrange the disulfide bonds of trypsin inhibitors toachieve a reduction in trypsin inhibitor activity.
 57. The process ofclaim 36 wherein a reducing agent is present during the concentrationand/or optional diafiltration step to disrupt or rearrange the disulfidebonds of trypsin inhibitors to achieve a reduction in trypsin inhibitoractivity.
 58. The process of claim 30 wherein a reducing agent is addedto the concentrated and optionally diafiltered soy protein solutionprior to drying and/or the dried soy protein product to disrupt orrearrange the disulfide bonds of trypsin inhibitors to achieve areduction in trypsin inhibitor activity. 59.-207. (canceled)